When sending chemical messages, cells release transmitters or hormones by Ca-triggered exocytosis. During exocytosis, the membrane of a secretory vesicle fuses with the plasma membrane. Fusion is mediated by three SNARE proteins and the proteins Munc18 and Synaptotagmin, and regulated by accessory proteins including Munc13, CAPS, complexin, granulophilin and rabphilin. Partial or complete structures exist for the SNAREs, for Munc18 and complexin, and our biochemical and structural knowledge about all the above proteins is substantial. In live cells, exocytosis has been explored exclusively by assays that, in one way or another, report membrane fusion. Foremost among them is electrophysiology. However, electrophysiology cannot directly report steps that precede fusion. In absence of direct assay, it is still unknown how the proteins mentioned above interact with each other or with secretory vesicles in living cells. We plan to use high-resolution light microscopy to observe single secretory granules in endocrine cells, as well as the exocytosis-related proteins recruited and released by them. We focus on how secretory vesicles dock at the plasma membrane. Single docking sites are observed simultaneously with the docking vesicle and its exocytosis. We use a microscope that has been calibrated by single-molecule imaging in terms of molecules per granule or molecules per cell surface are. If necessary, imaging is combined with electrophysiology. The goal is to understand the molecular steps in docking, and to determine which proteins form the molecular bridge connecting the membranes of a granule with that of the cell when docking has occurred. This work will provide a basic understanding of the role of each of the proteins of interest. More broadly, the work will explore an important and novel avenue to explore the action of proteins in living cells, at the level of single organelles and single proteins. Because most messages between cells are sent by exocytosis, exocytosis is an important cellular process. When exocytosis is defective in pancreatic beta cells then diabetes is the result. When exocytosis is defective or altered in neurons, then so is the basic mechanism of neural communication in the brain, and cognitive malfunctions result. Through increasing the basic mechanisms of exocytosis and its control, this work will be helpful in finding treatment strategies for diseases where cells fail to send messages. PUBLIC HEALTH RELEVANCE: Most messages between cells are sent by exocytosis. Exocytosis requires a cascade of protein interactions. When any member of this cascade is deficient, impaired or inappropriate exocytosis will result. For instance, defective exocytosis in pancreatic beta can cause diabetes, and defective exocytosis in neurons will disrupt the basic mechanism of neural communication in the brain. By high resolution light microscopy in live cells, this project seeks to understand how individual proteins in the cascade contribute to the mechanism of exocytosis.